[Note: Literature references for the following background information and on conventional test methods and laboratory procedures well known to the ordinary person skilled in the art, and other such state-of-the-art techniques as used herein, are indicated by reference numbers in parenthesis and appended at the end of the specification.]
Oxidative modification of biomolecules may contribute to the pathogenesis of disorders ranging from atherosclerosis to diabetes to ischemia-reperfusion injury (1-3). It also may play a role in the aging process itself (4,5). Important targets for oxidation include proteins, which play fundamental roles as biological catalysts, gene regulators, and structural components of cells (6-9). A well-characterized pathway for generating oxidants involves the NADPH oxidase of phagocytes. This membrane-associated electron transport chain produces superoxide (O.sub.2.sup..cndot.-) (10). EQU NADPH+O.sub.2 .fwdarw.NADP.sup.+ +O.sub.2.sup..cndot.-
The superoxide then dismutates to form hydrogen peroxide (H.sub.2 O.sub.2), which serves as an oxidizing substrate for myeloperoxidase, a heme protein secreted by activated phagocytes (6-9).
The NADPH oxidase system plays a key role in host defenses against microbial pathogens, and its importance is illustrated by clinical features of chronic granulomatous disease (CGD). In this genetic disorder, defects in components of the oxidase impair O.sub.2.sup..cndot.- production, rendering patients vulnerable to recurrent bacterial and fungal infections (6-9,11,12). Mice made deficient in NADPH oxidase simulate the CGD phenotype (13).
Although oxidants generated by phagocytes are critical to host defenses, they may also damage tissue at sites of inflammation (1,14). It has been difficult to evaluate their pathogenic roles, however, because many of the current methods are nonspecific and prone to artifacts. In contrast, analyzing normal and diseased tissue for specific markers has proved to be a powerful approach to studying oxidative damage in vivo (15). Such markers have been identified in vitro by searching for stable products of protein oxidation. For example, oxidatively damaged proteins may contain o,o'-dityrosine cross-links, which can be generated through a variety of mechanisms. These may involve peroxynitrite, nitrogen dioxide radical, hydroxyl radical, or the myeloperoxidase-peroxide system of activated phagocytes, which cross-links tyrosine residues (16-19). Tissue levels of o,o'-dityrosine are elevated in atherosclerosis, exercise, Alzheimer's disease, and aging (20-23). Oxidative stress has been linked to all of these conditions.
Dityrosine also may mark oxidatively damaged proteins for proteolytic destruction because red blood cells exposed to H.sub.2 O.sub.2 release this abnormal amino acid, suggesting that its formation in proteins may lead to protein breakdown (24).
The present inventor has proposed that oxidized amino acids might be excreted from cells rather than being re-used for protein synthesis. After entering the blood, they would be filtered by the kidney into urine. The demonstrations in the priority applications, Ser. No. 60,074,167 and 09/170,513, that levels of urinary o,o'-dityrosine are elevated in exercised and aging animals and that antioxidant therapy decreases these levels, is consistent with this proposal (21,22).